The present invention relates to a novel isoxazoline derivative, pharmaceutically acceptable salts, esters and stereochemically isomeric forms thereof which can serve as an inhibitor for protein caspases (cysteinyl-aspartate proteinases), a process for preparing the same and the use of the derivative as an inhibitor for caspases. The present invention also relates to a pharmaceutical composition for preventing inflammation and apoptosis which comprises the isoxazoline derivative, pharmaceutically acceptable salts, esters and stereochemically isomeric forms thereof and the process for preparing the same. The isoxazoline derivative according to the present invention can effectively be used in treating diseases due to caspases, for example, the disease in which cells are abnormally died, dementia, cerebral stroke, AIDS, diabetes, gastric ulcer, hepatic injure by hepatitis, sepsis, organ transplantation rejection reaction and anti-inflammation.
All organisms in nature undergo the life cycle consisting of development, differentiation, growth and death. Recently, an extensive research has been made to a mechanism involved in apoptosis which would play a key role in the control of the life cycle and the outbreak of diseases. It has been reported that apoptosis is occurred by a number of factors, but largely due to three kinds of cellular signal transport systems: the first of which is a signal transport system by the protein-protein interaction (See, Muzio M. et al., Cell 85, 817, 1996; Humke E. W. et al., JBC 273, 15702, 1998), the second, an incorporation of cytochrome C into cytoplasm via mitochondria (See, Liu X. et al., Cell 86:147, 1996; Li P. et al., Cell 91, 479, 1997), and the third, a cellular signal transport pathway by the SAPK(Stess-activated protein kinases) activation of mitogen-activation protein kinase(MAPK) enzymes. AU the pathways have been known to activate caspases cascade. As such caspases, about 10 kinds of isoenzymes in human and 14 kinds in mouse have been identified (see, Thornberry N. A. et al., Science 28, 1312 1998; Green D. R. Science 28, 1309, 1998; Ahmad A, et al., Cancer Res. 15, 5201 1998). The enzymes exist within the cells in the form of proenzyme which has no enzymatic activity and converted into an activated form if the cells are damaged or are exposed to a substance which leads to cellular necrosis. An activated enzyme has a heterodimer structure in which two polypeptides, i.e. larger subunits with the molecular weight of about 17-20 kDa, and smaller subunits with the molecular weight of about 10 kDa are bound together.
At present, caspases are classified into three (3) groups in view of the genetic identification analysis results and the biochemical characteristics: the first group is caspase-1, 4 and 5 which are responsible for the processing of cytokine activation, the second is caspase-3, 6 and 7 which carry out apoptosis and the third is caspase-8, 9 and 10 which are responsible for enzymatic activation in the upstream of signal transport system of apoptosis.
Among these caspases, Caspase-3 group and Caspase-8, 9, 10 etc. were recently reported to be related to apoptosis, and diseases (see, Thornberry N. A. et al., Science, 28, 1312, 1998).
According to the recent research results, caspases are commonly activated as apoptosis is initiated, even though there is a minor difference depending upon the tissues and cells. The activated caspases then activate intracellular CAD(Caspase-activated DNAse) which finally digests intranuclear DNA to result in cell death (Sakahira H., et al., Nature 1 96, 1998; Enari M et al., Nature 1 43, 1998). In addition, they promote apoptosis by decomposing substrate such as PARP (Poly-ADP ribose polymerase) which is necessary for the survival of cells.
Meantime, according to the recent disease-related researches, it was reported that the activity of Caspase-3 is increased in the brain of dementia patient which promotes the production of beta-amyloid peptide from beta-amyloid precursor protein that is considered to be a major cause of dementia, thereby accelerating the apoptosis of brain cells (see, Kuida K. et al., Nature 28, 368, 1996). Further, it was reported that activation of caspases can be the direct inducer of various diseases such as sepsis (see, Haendeler J. et al., Shock 6, 405, 1996; Lenhoff R J. et al., 29, 563, 1999), rheumatoid arthritis (Firestein G. S. et al., J. Clin Invest 96(3), 1631, 1995), cerebral stroke (see, Hill I. E. et al., Brain Res. 10, 398, 1995), ALS disease (see, Alexianu M. E. et al., J. Neurochem 63, 2365, 1994), autoimmune isease (see, Rieux-Laucat F, et al., Science 2, 1347, (1995), diabetes mellitusd(see, Juntti-Berggren et al., Science 2, 86, 1993), hepatitis (Haendeler J. et al., Shock 6, 405, 1996), organ transplantation rejection reaction (Koglin J. et al., Transplantation, 27, 904, 1999; Bergese S. D. et al., Transplantation 27, 904, 1999), gastric ulcer (see, Slomiany B. L. et al., J. Physiol. Pharmacol. 96, 1631, 1995), and the like.
The researches on three dimensional structure of caspase-1 and caspase-3, catalytic mechanism of the enzyme and enzyme-substrate specificity (see, Wilson, K. P et al., Nature 370, 270, 1994; Walker, N. P. C. et al., Cell 78, 343, 1994; Nature Struc. Biol. 3, 619, 1996) revealed that Caspase-1 group has a hydrolase-substrate specificity for the peptide sequence of (P4)-Val-X-Asp(P1) and Caspase-3 group has a hydrolase-substrate specificity for the sequence of (P4)Asp-X-X-Asp(P1).
Z-VAD-fluoromethyl ketone, and Z-DEVD-fluoromethyl ketone which mimics the above amino acid sequence have already been used in the researches on the inhibitors and were proven to have an inhibitory activity on apoptosis of hepatic cells by an activation of caspases (see, Rodriguez I. Et al., J. Exp. Med., 184, 2067, 1996; Rouquet N. et al., Curr Biol. 1, 1192, 1996; Kunstle G. et al., Immunol. Lett 55, 5, 1997), and on the apoptosis of brain cells by cerebral ischemias. However, since such peptide derivatives are deficient in drug property for clinical application, they cannot be used as therapeutics.
Fulminant hepatic failure (FHF) is a clinical syndrome resulting from massive death of liver cells or sudden and severe impairment of liver function (See: Trey, C. et al., 1970, Progress in liver disease, Popper, H. and F. Schaffner, eds. Grune and stratton, New York, pp282-298). The causes of FHF are diverse: hepatitis virus infection, drugs and toxins, alcohol, ischemia, metabolic disorder, massive malignant infiltration, chronic autoimmune hepatitis, etc. However, these mechanisms are not completely clear. Since the prognosis of FHF is very poor while its progress is very rapid, it is not uncommon that a patient falls in lethal condition in 1-2 weeks from the onset of this syndrome (See, Sherlock, S. 1993, Adv. Intern. Med. 38: 245-267). Consequently, the overall mortality in most series is very high. However, the hepatic lesion is potentially reversible, and survivors usually recover completely.
Different therapeutic options that have been tried in FHF include antibiotics, diuretics, corticosteroids, blood transfusion, charcoal haemoperfusion, and plasmaphresis. However, none of these methods have been shown to be effective in controlled studies. In recent years, liver transplantation is generally accepted as the only therapeutic option to actually improve the prognosis of this syndrome. However, liver transplantation cannot be the perfect treatment for FHF because of immune complication, viral or bacterial infection, and graft availability. Thus, a potent therapeutic agent which can protect hepatic cells from massive death during the acute phase is critically desired.
Apoptosis is a type of cell death characterized by a series of distinct morphological and biochemical changes accomplished by specialized cellular machinery. Apoptosis is an essential process to remove excess, unwanted and harmful cells and maintain homeostasis, but inappropriate apoptosis is implicated in many human diseases such as neurodegenerative diseases, ischaemic damage, autoimmune disorders, several forms of cancer. Recently, it became clear that apoptosis of hepatocytes is a critical cause of hepatic injury in viral hepatitis and alcoholic hepatitis and acute hepatic failure in fulminant hepatitis. Many changes which occur in a cell that received apoptotic signal reflect complex biochemical events carried out by a family of cysteine proteases called caspases.
Caspases inactivate proteins that protect living cells from apoptosis, such as ICAD/DFF45, an inhibitor of the nuclease responsible for DNA fragmentation, and Bcl-2. At the same time, caspases contribute to apoptosis not only by direct disassembly of cell structures, but also by reorganizing cell structures indirectly by cleaving several proteins involved in cytoskeleton regulation. Since caspase activation is closely related to the initiating, propagating, and terminal event of most forms of apoptosis, this family of enzymes are attractive potential targets for the treatment of disorders resulted from excessive apoptosis or insufficient apoptosis.
Several kinds of caspase inhibitors have been identified. Four distinct classes of viral inhibitors have been described: CrmA, p35, a family of IAP (inhibitors of apoptosis), and the hepatitis B virus-encoded HBx protein (See, Gottlob, K et al., 1998, J. Biol. Chem. 273: 33347-33353). However, these molecules are not suitable as the therapeutic agent. Peptide-based caspase inhibitor such as z-VAD-fmk, z-DEVD-fmk, and Ac-YVAD-cmk has widely been used for research use and this inhibitor showed apoptosis-blocking activity in cellular level (See: Sane, A. T. et al., 1998, Cancer Res. 58: 3066-3072), in rodent models of liver injury caused by. Fas or by TNFxcex1 (See: Kunstle, G. et al., 1997, Immunol. Lett. 55; 5-10) or ischemia after liver transplantation (See: Cursio, R. et al., 1999, FASEB J. 13: 253-261). Petak and colleagues showed that a bi-functional anticancer agent, BCNU (1,3-bis(2-chloroethyl)-1-nitrosourea) had caspase inhibiting activity and inhibited drug-induced apoptosis in vitro (See: Petak, I. et al., 1998, Cancer Res. 58: 614-618). Recently, cyclooxygenase-2 (COX-2) inhibitors are arousing interest as potential therapeutic agents of FHF (See, McCormick, P. A. et al., 1999, Lancet 353: 40-41). However, the efficacy of these materials has not been clinically verified yet.
In the meantime, development of new drugs depends primarily on the availability of suitable animal models relevant to human hepatitis or hepatocytic damage. It is therefore very important to adopt a suitable animal model relevant to human FHF to test efficacy of a candidate for therapeutic agent. Two types of experimental hepatitis model were reported. One is hepatic injury induced by bacterial lipopolysaccharide together with D-galactosamine (See: Galanos, C. et al., 1979, Proc. Natl. Acad Sci., 76: 5939; Lehman, V. et al., 1987, J. Exp. Med. 165-657), and the other is a recently developed experimental model, Con A-induced hepatitis (See: Tiegs, G. et al., 1992, J. Clin. Invest. 90: 196-203; Mizuhara, H. et al., 1994, J. Exp. Med. 179: 1529-1537). Con A-induced hepatitis model closely mimics human FHF in many respects, especially in the role of Fas in pathogenesis. Fas is abundantly expressed on the hepatocyte and FasL is expressed on activated T cells and functions as an effector of cytotoxic lymphocytes. Injection of agonistic monoclonal anti-Fas antibody into adult mice caused rapid hepatic failure, indicating that abnormally activated Fas-FasL system may play a role in human fulminant hepatitis which can be caused by the activation of immune system such as cytotoxic T cells. Accumulating data such as the involvement of specific CTLs in the pathogenesis of FHF, the sensitivity of primary hepatocytes to Fas-mediated apoptosis in vitro, and the overexpression of Fas in hepatocytes transformed with human hepatitis virus are consistent with tis hypothesis. In recent studies, the activation of Fas-FasL system has been proved to play an important role in the liver cell injury by Con A-induced hepatitis (See: Tagawa, Y. et al., 1998, Eur. J. Immunol. 28: 4105-4113). FasL was induced in the liver shortly after the Con A injection was predominantly expressed on intrahepatic T cells. These results indicate thar Fas-FasL system is a critical element in the development of Con A-induced hepatitis. At the same time, the induction of Con A-hepatitis is associated with the production of various cytokines such as IL-2, TNFxcex1, IL-6, IL-4, and IL-10.
The present inventors have conducted an extensive research for many years in order to develop new therapeutics suitable for caspase inhibitor which has a unique structure over those known in the art. As a result the inventors have surprisingly discovered a novel isoxazoline derivative of formula (I) which has a different structure over the known inhibitors and has an excellent inhibitory activity against various substrates for caspases, and have completed the present invention
It is therefore an object of the present invention to provide a novel isoxazoline compound of the formula (I), the pharmaceutically acceptable salts, esters and stereochemically isomeric forms thereof which are useful as a caspase inhibitor.
Another object of the present invention is to provide a process for preparing the compound of formula (I).
Further object of the present invention is to provide a caspase inhibitor which comprises an isoxazoline derivative of the formula (I), the pharmaceutically acceptable salts, esters and stereochemically isomeric forms thereof.
Still other object of the present invention is to provide a pharmaceutical composition for inhibiting caspases activity which comprises as the active ingredient a therapeutically effective amount of the isoxazoline derivative of formula (I) and pharmaceutically acceptable carrier.
Still further object of the present invention is to provide a pharmaceutical composition for preventing inflammation and apoptosis which comprises the isoxazoline derivative, pharmaceutically acceptable salts, esters and stereochemically isomeric forms thereof and the process for preparing the same.
Further objects and advantages of the invention will become apparent through reading the remainder of the specification.
The foregoing has outlined some of the more pertinent objects of the present invention These objects should be construed to be merely illustrative of some of the more pent features of the invention. Many other beneficial results can be obtained by applying the disclosed invention in a different manner or by modifying the invention within the scope of the disclosure. Accordingly, other objects and a more thorough understanding of the invention may be found by referring to the detailed description of the preferred embodiment in addition to the scope of the invention defined by the claims.